• Journal of Life Science
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• v.29 no.12
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• pp.1408-1414
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• 2019

Enzymes are widely used in industrial applications such as detergents, food, feed production, pharmaceuticals and medical applications and major contributors to clean industrial products and processes. To screen, identify, and characterize the enzymes the zymography techniques are routinely used. The zymography technique is a simple, sensitive, and quantifiable technique that is widely used to detect functional enzymes following electrophoretic separation in sodium dodecyl sulfate (SDS)-polyacrylamide gels. The method is a versatile two-stage technique involving protein separation by electrophoresis followed by the detection of enzyme activity in polyacrylamide gels under non-reducing conditions. It is based on SDS-polyacrylamide gel (PAG) copolymerization with substrates, which are degraded by the hydrolytic enzymes restored in enzyme reaction buffer after the electrophoretic separation. Any kind of biological sample can be applied and analyzed on zymography, including culture supernatants of microbes, plants extracts, blood, tissue culture fluids, enzymes in foods extracts and metaproteome. The advantage of zymography is that it is possible to directly detect the protein with activity on the electrophoretic gel as well as confirm the activity at the nanogram level. Thus, this zymography technology can be applied in various fields. However, these advantages are rather disadvantageous and can often lead to experimental errors. In this review, the advantages, disadvantages, and problem solving of zymography technique are described.

• Korean Journal of Microbiology
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• v.51 no.3
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• pp.187-193
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• 2015

Candida (also known as Pseudozyma) antarctica lipase B (CAL-B) has been intensely studied in academic and industrial fields. However, the research related to its homologous enzymes has been rarely reported. In the current investigation, protein sequence similarity search of CAL-B has been conducted and six homologous protein sequences were identified. After the syntheses of their codon-optimized genes, the synthetic genes have been cloned into a periplasmic expression vector to express in Escherichia coli. Among six homologous sequences, four sequences were successfully expressed in E. coli. The hydrolytic activities of the expressed proteins towards 4-nitrophenyl acetate and 4-nitrophenyl butyrate were measured and compared with those of CAL-B to identify whether the expressed proteins work as a hydrolase. It has been revealed that the expressed proteins can hydrolyze the substrates and the specific activities were determined as $(1.3-30){\times}10^{-2}{\mu}mol/min/mg$, which are lower than those of CAL-B. Among these homologous enzymes, Pseudozyma hubeiensis SY62 exhibits the comparable enantioselectivity to that of CAL-B towards the hydrolysis of (${\pm}$)-1-phenylethyl acetate.

• Journal of Microbiology and Biotechnology
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• v.24 no.6
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• pp.771-780
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• 2014

A putative lipolytic enzyme gene, named as est9x, was obtained from a marine microbial metagenome of the South China Sea. Sequence analysis showed that Est9X shares lower than 27% sequence identities with the characterized lipolytic enzymes, but possesses a catalytic triad highly conserved in lipolytic enzymes of the ${\alpha}/{\beta}$ hydrolase superfamily. By phylogenetic tree construction, Est9X was grouped into a new lipase/esterase family. To understand Est9X protein in depth, it was recombinantly expressed, purified, and biochemically characterized. Within potential hydrolytic activities, only lipase/esterase activity was detected for Est9X, confirming its identity as a lipolytic enzyme. When using p-nitrophenol esters with varying lengths of fatty acid as substrates, Est9X exhibited the highest activity to the C2 substrate, indicating it is an esterase. The optimal activity of Est9X occurred at a temperature of $65^{\cric}C$, and Est9X was pretty stable below the optimum temperature. Distinguished from other salt-tolerant esterases, Est9X's activity was tolerant to and even promoted by as high as 4 M NaCl. Our results imply that Est9X is a unique esterase and could be a potential candidate for industrial application under extreme conditions.

• Nutrition Research and Practice
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• v.8 no.3
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• pp.278-283
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• 2014

BACKGROUND/OBJECTIVES: Due to its beneficial health effects, use of buckwheat has shown a continuous increase, and concerns regarding the allergic property of buckwheat have also increased. This study was conducted for evaluation of the hydrolytic effects of seven commercial proteases on buckwheat allergens and its allergenicity. MATERIALS/METHODS: Extracted buckwheat protein was hydrolyzed by seven proteolytic enzymes at individual optimum temperature and pH for four hours. Analysis was then performed using SDS-PAGE, immunoblotting, and competitive inhibition ELISA (ciELISA) with rabbit antiserum to buckwheat protein, and direct ELISA with pooled serum of 21 buckwheat-sensitive patients. RESULTS: Alkaline protease, classified as serine peptidase, was most effective in reducing allergenicity of buckwheat protein. It caused decomposition of the whole buckwheat protein, as shown on SDS-PAGE, and results of immunoblotting showed that the rabbit antiserum to buckwheat protein no longer recognized it as an antigen. Allergenicity showed a decrease of more than 50% when pooled serum of patients was used in ELISA. Two proteolytic enzymes from Aspergillus sp. could not hydrolyze buckwheat allergens effectively, and the allergenicity even appeared to increase. CONCLUSIONS: Serine-type peptidases appeared to show a relatively effective reduction of buckwheat allergenicity. However, the antigenicity measured using rabbit antiserum did not correspond to the allergenicity measured using sera from human patients. Production of less allergenic buckwheat protein may be possible using enzymatic hydrolysis.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.5
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• pp.382-390
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• 2006

Various microorganisms were isolated from the surface waters and sediments of eutrophic lakes and reservoirs in Korea to enable an investigation of bacteria having algal lytic activities against Anabaena flos-aquae when water blooming occurs and to study enzyme profiles of algal lytic bacteria. Two bacterial strains, AFK-07 and AFK-13, were cultured, characterized and identified as Acinetobacter johnsonii and Sinorhizobium sp., respectively. The A. johnsonii AFK-07 exhibited a high level of degradatory activities against A. flos-aquae, and produced alginase, caseinase, lipase, fucodian hydrolase, and laminarinase. Moreover, many kinds of glycosidase, such as ${\beta}-galactosidase,\;{\beta}-glucosidase,\;{\beta}-glucosaminidase,\;and\; {\beta}-xylosidase$, which hydrolyzed ${\beta}-O-glycosidic$ bonds, were found in cell-free extracts of A. johnsonii AFK-07. Other glycosidases such as ${\alpha}-galactosidase,\;{\alpha}-N-Ac-galactosidase,\;{\alpha}-mannosidase,\; and\;{\alpha}-L-fucosidase$, which cleave ${\alpha}-O-glycosidic$ bonds, were not identified in AFK-07. In the Sinorhizobium sp. AFK-13, the enzymes alginase, amylase, proteinase (caseinase and gelatinase), carboxymethyl-cellulase (CMCase), laminarinase, and lipase were notable. No glycosidase was produced in the AFK-13 strain. Therefore, the enzyme system of A. johnsonii AFK-07 had a more complex mechanism in place to degrade the cyanobacteria cell walls than did the enzyme system of Sinorhizobium sp. AFK-13. The polysaccharides or the peptidoglycans of A. flos-aquae may be hydrolyzed and metabolized to a range of easily utilized monosaccharides or other low molecular weight organic substances by strain AFK-07 of. A. johnsonii, while the products of polysaccharide degradation or peptidoglycans were more likely to be utilized by Sinorhizobium sp. AFK-13. These bacterial interactions may offer an alternative effective approach to controlling the water choking effects of summer blooms affecting our lakes and reservoirs.

• Microbiology and Biotechnology Letters
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• v.18 no.4
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• pp.437-441
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• 1990

For the genetic development of more powerful antagonistic Pseudomom - YPL-1 as a biocontxol agent against soilborne plant pathogenic Fuaarium solani causing root rot of many important crops, mutants improving the productivity of chitinase were obtained by mutation with UV radiation or NTG treatment, P. stutzeri YPL-M26 (UV mutant) and P. stutzeri YPL-MI78 (NTG mutant) could improve the productivity of chitinase by 2.5 and 2.0 times, and its antifungal activity by 1.7 and 1.5 times, respectively. The antifungal mechanism of P. stutzeri YPL-M26 was caused by lysis of the fungal cell wall by hydrolytic enzymes such as chitinase. The antifungal activity of crude chitinase of P. stutzeri YPLM26 on the mycelial growth of F. solani was observed to be much higher than that of the original strain. The enzymes produced by P. stutzeri YPL-M26 were the same as the original strain in enzymatic properties such as optimal pH and temperature.

• Polymer(Korea)
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• v.29 no.1
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• pp.54-58
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• 2005

The hydrolytic behavior of microbial poly[(R)-3-hydroxybutyrate]](P(3HB)) has been studied by using two model systems, Langmuir monolayer and solution-grown single crystals (SCs), for elucidating the mechanism for both alkaline and enzymatic degradations. An initial degradation of SCs of P(3HB) leads to breakup lamellae parallel to their short axis (b-axis). Similarly, ridge formation on the lamellar surface appears along the b-axis at lower quenching temperature than melting temperature. Both results support that the lamellar crystals contain less-ordered and more thermally sensitive regions along the b-axis. Although the enzymatic hydrolysis of P(3HB) monolayers was similar to its alkaline one, the enzymatic degradation of P(3HB) monolayers occurred at higher constant surface pressure than the alkaline degradation. This behavior might be attributed to the size of enzymes which is much larger than that of alkaline ions; that is, the enzymes need larger contact area with monolayers to be activated.

• Natural Product Sciences
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• v.16 no.1
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• pp.1-5
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• 2010

$\beta$-Glucuronidases (E.C. 3.2.1.31) from Escherichia coli, Helix pomatia, and bovine liver activity have been investigated on 7-O-glucuronides (baicalin, wogonoside, and luteolin-7-O-glucuronide) and 3-O-glucuronides (quercetin-3-O-glucuronide and kaempferol-3-O-glucuronide). Bovine liver enzyme was not active on any of these substrates. E. coli and H. pomatia enzymes were active on 7-O-glucuronides, however, 3-O-glucuronides were resistant to $\beta$-glucuronidase hydrolysis. These results suggest that glucuronic acid at 7-position is more susceptible to E. coli and H. pomatia $\beta$-glucuronidases than that at 3-position. In addition, the subtle difference of aglycone structure on 7-O-glucuronides affected the preference of enzyme. E. coli enzyme was favorable for the hydrolysis of baicalin, however, H. pomatia enzyme was found to be efficient for the hydrolysis of wogonoside. Both enzymes showed the similar hydrolytic activity towards luteolin-7-O-glucuronide. When the Scutellaria baicalensis crude extract was subjected to enzymatic hydrolysis, baicalin and wogonoside were successfully converted to their aglycone counterparts with H. pomatia at 50 mM sodium bicarbonate buffer pH 4.0. Accordingly, the enzymatic transformation of glycosides may be quite useful in preparing aglycones under mild conditions.

• Korean Journal of Food Science and Technology
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• v.53 no.6
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• pp.695-700
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• 2021

The purpose of this study was to prepare low-molecular weight collagen using a commercial proteolytic enzyme (Protamex) from collagen extracted from feet of Korean native chicken and to investigate the quality characteristics of this collagen. The collagen content of Korean native chicken feet was 13.9 g/100 g, which was higher than the 6.21 g/100 g of general broilers. It was found that the content of low molecular weight collagen increased as the concentration of proteolytic enzymes and reaction time increased. In particular, reaction with 1% Protamex for 7 h resulted in 55.6% of low molecular weight (1,000-5,000 Da) collagen content, and the average molecular weight was 5,390 Da. Regarding the texture of the enzyme-treated collagen, the collagen with high molecular weight peptides decomposed into low molecular weight peptides, and the gel type could not be formed, whereas the sol type was maintained.

• The Korean Journal of Food And Nutrition
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• v.36 no.1
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• pp.42-49
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• 2023

To enhance the efficacy of Abeliophyllum distichum leaves, extracts were prepared using different solvents for hydrolytic enzyme-treated Abeliophyllum distichum leaves. Physicochemical quality and antioxidant activity were measured. Soluble solids, reducing sugar, ascorbic acid, flavonoids, and polyphenols contents showed the lowest values in the control without enzyme treatment. However, they showed high contents in ethanol extract. In the case of enzyme treatment, their values were higher than those of the control. In particular, verbascoside content increased about 220 times more than that of the control group when treated with enzymes and extracted with 50% ethanol. pH was lowered upon enzymatic treatment. Regarding DPPH radical scavenging activity, for enzyme-free, 25% ethanol extract showed the highest activity among extracts with different solvents. For cellulase and pectinase-treated leaves, water extract showed the highest DPPH radical scavenging activity among extracts with different solvents. For leaves treated with enzyme combination, 50% ethanol extract showed the highest DPPH radical scavenging activity among extracts with different solvents. Regarding ABTS radical scavenging activity, it was generally higher in the 50% ethanol extract than in the water extract and 25% ethanol extract. In particular, verbascoside content was increased when the extract was prepared by co-treatment with enzymes and 50% ethanol.